PROJECT SUMMARY Human induced pluripotent stem cells (iPSCs) provide a promising source of patient-specific cardiac cells. Our team has pioneered development of protocols to differentiate iPSCs to cardiomyocytes (iPSC-CMs) but these cells lack mature, adult-like phenotypes. One hallmark of CM maturation is a transition from glycolysis and glucose oxidation to fatty acid oxidation as the dominant metabolic pathway, among other metabolic changes. Our premise is that identifying metabolic critical quality attributes (CQAs) of maturity will provide fundamental insight into phenotypic maturation of iPSC-CMs, new tools to facilitate discovery of effective strategies to mature iPSC-CMs, and novel technologies to monitor maturation state during iPSC-CM biomanufacturing. To achieve this premise, we will employ an integrative quantitative metabolomics and proteomics approach to profile metabolite and metabolic enzyme concentrations, and metabolic pathway utilization, in iPSC-CMs undergoing maturation by extended time in culture or biochemical/biomechanical stimulation. Comparing metabolic transitions during in vitro iPSC-CM maturation to metabolic transitions during development in vivo and to acquisition of maturation phenotypes will allow us to map metabolic transitions to developmental processes. We will perform multivariate data analyses to predict metabolic CQAs of maturation phenotypes and build novel tools to monitor these CQAs during iPSC-CM biomanufacturing. Thus, the proposed study will provide fundamental new insight into metabolic pathway utilization during iPSC-CM maturation and cardiac development, and will predict metabolic CQAs that will facilitate monitoring progression of maturation in iPSC-CMs during biomanufacturing. Our specific aims are: 1. Profile metabolic transitions during iPSC-CM differentiation and maturation. At different iPSC-CM maturation stages induced by extended culture, micropatterned substrates, carbon source availability, and electromechanical stimulation, we will quantify metabolite and protein concentrations via metabolomics and proteomics and targeted metabolic assays. We will assess maturation via molecular and functional assays to relate changes in metabolites and metabolic pathway utilization to acquisition of maturation phenotypes. 2. Assess metabolic pathway enrichment in developing murine cardiomyocytes. We will use metabolomics and proteomics to profile metabolite and protein concentrations in murine CMs at different developmental stages and compare to metabolic transitions that occur during iPSC-CM maturation. 3. Identify metabolic CQAs and develop tools for assessment of iPSC-CM maturity during biomanufacturing. We will use multivariate analysis to predict metabolic CQAs that identify maturation state of iPSC-CMs. We will then develop assays to monitor these CQAs (combinations of metabolite and metabolic enzymes) during biomanufacturing via targeted metabolomics/proteomics, and by use of CRISPR-Cas9 gene editing to engineer reporters of metabolic pathway transitions that mark iPSC-CM maturation states.